Partner of SLD five 1 (PSF1)-derived peptide

ABSTRACT

A Partner of Sld Five 1 (PSF1)-derived peptide that can induce a Cytotoxic T-Cell (CTL) response useful for a specific immunotherapy for cancer patients and a pharmaceutical composition for treating or preventing cancer, which contains the peptide according to the present invention are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of, and claims the benefitof, co-pending U.S. patent application Ser. No. 15/413,343, filed onJan. 23, 2017, which is a continuation of Ser. No. 14/424,981, filed onFeb. 27, 2015, which is a national phase application under 35 U.S.C. §371 of International Application No. PCT/JP2013/073084, filed on Aug.29, 2013, which claims priority under 35 U.S.C. § 119 to Japanese PatentApplication No. 2012-191050, filed on Aug. 31, 2012, the contents ofwhich are hereby incorporated by reference in their entirety for allpurposes.

TECHNICAL FIELD

The present invention relates to PSF1-derived peptides which are usefulfor the specific immunotherapy for Human Leucocyte Antigen-A02 (HLA-A02)positive cancer patients.

BACKGROUND OF THE DISCLOSURE

PSF1 forms tetramer, go-ichi-ni-san (GINS) complex, with SLD5, PSF2 andPSF3 of constitutive protein, which binds to MCM (mini-chromosomemaintenance complex) and cell division cycle 45 (cdc45). That is knownto be associated with initiation of DNA replication and elongation(non-patent reference 1: Structure of the human GINS complex and itsassembly and functional interface in replication initiation. Kamada etal. Nat. Struct. Mol. Biol. 2007; 14:388-396 and non-patent reference 2:The human GINS complex associates with cdc45 and MCM and is essentialfor DNA replication. Aparicio et al. Nucleic Acid Res. 2009;37:2087-2095).

With respect to the relation with cancer, the expression of PSF1 isconsidered to increase in human breast cancer cell. It is also reportedthat the reduction of the expression of PSF1 had caused the suppressionof proliferation significantly. Furthermore, it is reported that overallsurvival rate among breast cancer patients, of which the PSF1 expressionlevel is low in its cancer tissue, is significantly higher, compared tothe patients, of which the level is high (non-patent reference 3:Up-regulation of psf1 promotes the growth of breast cancer cells. Izumiet al. Genes to Cells 2010; 15:1025-1024).

Expression of PSF1 has been documented in the cancer tissue derived frommalignant melanoma, lung cancer and esophagus cancer (Non-patentreference 4: Comprehensive expression profiling of tumor cell linesidentifies molecular signatures of melanoma progression. Ryu B. et al.ProsOne 2007; 7:e594, Non-patent reference 5: PSF1, a DNA replicationfactor expressed widely in stem and progenitor cells, drives tumorigenicand metastatic properties. Nagahama Y. et al. Cancer Res. 2010;70:1215-24, and Patent document 1: International Publication GazetteWO2003/42661). The relation between PSF1 and the cancer stem cell isalso known in non-patent reference 5.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a peptide which isuseful for the specific immunotherapy for cancer patients. Our inventorsfound PSF1-derived peptides, which is associated with the extracellulardomain of HLA-A02 molecule on human breast cancer cell strain. And theyconfirmed that these peptides had induced a peptide specific CTL(cellular cytotoxicity T-cell) and achieved the present invention.

In accordance with one aspect of the present invention, there isprovided a peptide having CTL inducibility, where the peptide is derivedfrom an amino acid sequence of SEQ ID NO: 1. In this embodiment, thepeptide includes the continuous 8-14 base within 70-110 of SEQ ID NO.:1.

In accordance with a second aspect of the present invention, there isprovided a peptide having CTL inducibility that includes an amino acidsequence of SEQ ID NO.: 3, 4, or 5.

In accordance with a third aspect of the present invention, there isprovided a peptide consisting of an amino acid sequence of SEQ ID NO.:3, 4, or 5. In this embodiment, 1, 2, or several amino acids aresubstituted, deleted, or added in the amino acid sequence of SEQ ID NO.:3, 4, or 5.

According to the embodiments of the present invention, PSF1-derivedpeptides having inducibility of CTL, which can kill tumor cells inHLA-A02⁺ cancer patients, are provided. Accordingly, the specificimmunotherapy for HLA-A02⁺ cancer patients becomes possible.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure is described in conjunction with the appendedfigures:

FIG. 1 shows a result of detection of a peptide specific CTL by tetramerstaining. CTL cells obtained by incubation were subjected to doublestaining with PE-labeled tetramer and APC-H7-labeled anti-CD8 antibodiesso that the fluorescent intensity was measured with a FACS Aria. Cellfractions stained with both of the labels were detected at positionsenclosed by lines (an upper portion in each panel). Since positive cellswere observed in two types of CTL lines (0209-01 H2 and 0209-02 D2), itwas found that CTL recognizing YLYDRLLRI (SEQ ID NO.: 3) was obtained.

FIG. 2A shows cellular cytotoxicity by a peptide specific CTL line.Peptide-pulsed cells (peptide+) or peptide-non-pulsed cells (peptide-)obtained from CTL specific to peptide YLYDRLLRI (SEQ ID NO.: 3) (E;effector cell) and T2 cells (T; target cell) were cocultured at an E:Tcell ratio of 1:1, 3:1, and 10:1. On the next day, the survival rate oftarget cells was obtained. Then, it was found that the survival rate oftarget cells pulsed with peptides decreased to a degree greater thantarget cells not pulsed with peptides. In addition, since the survivalrate decreased in accordance with the ratio of cocultured cells,cellular cytotoxicity by peptide specific CTL was demonstrated.

FIG. 2B shows cellular cytotoxicity by a peptide specific CTL line.Peptide-pulsed cells (peptide+) or peptide-non-pulsed cells (peptide-)obtained from CTL specific to peptide RALRWEYGSVLPN (SEQ ID NO.: 4) (E;effector cell) and T2 cells (T; target cell) were cocultured at an E:Tcell ratio of 1:1, 3:1, and 10:1. On the next day, the survival rate oftarget cells was obtained. Then, it was found that the survival rate oftarget cells pulsed with peptides decreased to a degree greater thantarget cells not pulsed with peptides. In addition, since the survivalrate decreased in accordance with the ratio of cocultured cells,cellular cytotoxicity by peptide specific CTL was demonstrated.

FIG. 2C shows cellular cytotoxicity by a peptide specific CTL line.Peptide-pulsed cells (peptide+) or peptide-non-pulsed cells (peptide-)obtained from CTL specific to peptide ALRWEYGSVL (SEQ ID NO.: 8) (E;effector cell) and T2 cells (T; target cell) were cocultured at an E:Tcell ratio of 1:1, 3:1, and 10:1. On the next day, the survival rate oftarget cells was obtained. Then, it was found that the survival rate oftarget cells pulsed with peptides decreased to a degree greater thantarget cells not pulsed with peptides. In addition, since the survivalrate decreased in accordance with the ratio of cocultured cells,cellular cytotoxicity by peptide specific CTL was demonstrated.

FIG. 3 shows the positions of YLYDRLLRI (SEQ ID NO.: 3), RALRWEYGSVLPN(SEQ ID NO.: 4), and ALRWEYGSV (SEQ ID NO.: 5) in an amino acid sequenceof PSF1 (SEQ ID NO.: 1). All the three peptides are located in theregion from 79 to 100 of PSF1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The ensuing description provides preferred exemplary embodiment(s) only,and is not intended to limit the scope, applicability or configurationof the disclosure. Rather, the ensuing description of the preferredexemplary embodiment(s) will provide those skilled in the art with anenabling description for implementing a preferred exemplaryembodiment(s) of the disclosure. It should be understood that variouschanges may be made in the function and arrangement of elements withoutdeparting from the spirit and scope of the invention as set forth in theappended claims.

Peptide and Polypeptide

The present invention provides a PSF1-derived peptide having CTLinducibility (hereafter, the peptide).

According to the embodiments of the present invention, “PSF1-derivedpeptide” refers to a peptide fragment consisting of an amino acidsequence which is a part of the amino acid sequence of PSF1 (SEQ ID NO.:1). The amino acid sequence (SEQ ID NO.: 1) and the nucleic acidsequence (SEQ ID NO.: 2) of PSF1 have been disclosed by GenBank underaccession number of NP_066545 and NM_021067, respectively.

The peptide of the present invention was discovered as peptides havinghigh binding affinity to the HLA-A02 antigens. This means that saidpeptide can form a complex with an HLA-A02 to be presented on the cellsurface.

According to the embodiments of the present invention, the phrase“having CTL inducibility” means that the peptide is recognized by aspecific CTL. In other word, the peptide has an ability to induce apeptide specific CTL. Although peptides having high binding affinity tothe HLA antigens as described above are expected to be highly effectiveas cancer vaccines, the candidate peptides, which can be available foran active pharmaceutical ingredient, must be examined for the actualpresence of CTL inducibility. The ability to induce the peptide specificCTL can be examined by, for example, determining whether or not acytokine such as interferon-γ (IFN-γ) is produced by peptide-stimulatedperipheral blood mononuclear cells (PBMCs) in response to antigenpresenting cells which are pulsed with said peptide using the ELISAtechnique or the like. In addition, cytotoxic activity of the inducedCTL can be determined by the ⁵¹Cr-release assay and the like. Thepeptide of the present invention is preferred to have 8 to 14, morepreferably 8 to 11 and especially, 9 or 10 amino acid residues in viewof good recognition by CTL.

As a result of examining the CTL inducibility of peptides as describedabove, it was discovered that peptides having high binding affinity toan HLA antigen did not necessarily have high inducibility. However,peptides selected from peptides comprising the amino acid sequencesindicated by YLYDRLLRI (SEQ ID NO.: 3), RALRWEYGSVLPN (SEQ ID NO.:4) andALRWEYGSV (SEQ ID NO.:5) showed particularly high CTL inducibility andcytotoxic activity. These all three peptides are located in the regionfrom 79 to 100 of PSF1 (FIG. 3). With respect to YLYDRLLRI (SEQ IDNO.:3), peptides including amino acid sequence thereof, AYLYDRLLRI (SEQID NO.:6) or YLYDRLLRIR (SEQ ID NO.: 7), also showed high CTLinducibility. Alternatively, with respect to ALRWEYGSV (SEQ ID NO.: 5),peptides including amino acid sequence thereof, ALRWEYGSVL (SEQ ID NO.:8) or RALRWEYGSV (SEQ ID NO.: 9), also showed high CTL inducibility.

Embodiments of the present invention also provide peptide derivatives ofa PSF1-derived peptide (hereafter, the peptide derivatives). Accordingto the embodiments of the present invention, “a peptide derivative of aPSF1-derived peptide” is a peptide consisting of the amino acid sequenceof the PSF1-derived peptide except that substitution, deletion and/orinsertion of one or two amino acids have been introduced and having CTLinducibility. Whether or not a peptide derivative has the desiredproperties can be determined by the above-described procedures.

In order not to alter the property of the original peptide, thesubstitution of an amino acid is preferably made within the amino acidsbelonging to the same family, such as polar amino acids, non-polar aminoacids, hydrophobic amino acids, hydrophilic amino acids, positivelycharged amino acids, negatively charged amino acids and aromatic aminoacids. The deletion and/or insertion of an amino acid are preferablymade so that the number of the amino acid residues consisting of thederivative is 8-14.

In general, peptides that are capable of binding to an Human LeucocyteAntigen (HLA) molecule shares some specific amino acid sequences withregularity depending on the types of the HLA. The specific amino acidsequences with regularity are called as “binding motifs”. That is, thebinding motif to an HLA-A02 is the sequence wherein the amino acid nextto the N-terminal is isoleucine, leucine, valine, methionine, alanine orthreonine and that at the C-terminal is isoleucine, leucine, valine,methionine, alanine or threonine (Current Pharmaceutical Design 2010,16:3149-3157). Binding of a peptide having the HLA-A02 binding motif toan HLA-A02 can be determined using computer analysis such asBioinformatics and Molecular Analysis Section (NIH, Bethesda, Md.)(Parker K C, et al., J. Immunol. 1994; 152:153-175).

According to the embodiments of the present invention, the substitution,deletion and/or insertion of an amino acid is preferably made so thatthe derivative is acceptable in view of an HLA binding motif. That is,the substitution, deletion and/or insertion of an amino acid ispreferably made so that the amino acid next to the N-terminal isisoleucine, leucine, valine, methionine, alanine or threonine and theC-terminus amino acid is isoleucine, leucine, valine, methionine,alanine or threonine.

The amino acids constituting the peptides and peptide derivatives of theinvention may be natural amino acids or amino acid analogues. Amino acidanalogues may include N-acylated, O-acylated, esterified, acid amidatedand alkylated amino acids. The amino or carboxylic group or the like ofthe amino acid residue constituting the peptide or peptide derivative ofthe invention may be modified so long as it does not significantlydeteriorate the function of the peptide. The modification may beaddition of formyl, acetyl or t-butoxycarbonyl group at the N-terminus-or free-amino group, or addition of methyl, ethyl, t-butyl or benzylgroup at the C-terminus- or free carboxylic group. Other modificationsmay include incorporation of D-amino acids or other amino acid mimeticsthat can be used, for example, to increase the serum halflife of thepeptides.

The peptide and peptide derivative according to the embodiments of thepresent invention may be synthesized by a conventionally used peptidesynthesizing procedure. For example, the peptides can be preparedsynthetically, using either recombinant DNA technology or chemicalsynthesis. Examples of the conventionally used procedures are thosedescribed in the literatures including “Peptide Synthesis”,Interscience, New York, 1966; “The Proteins”, vol. 2, Academic PressInc., New York, 1976.

Nucleic Acid and a Vector

The present invention further provides a nucleic acid molecule encodingthe peptide or peptide derivatives of the invention and a vectorcomprising said nucleic acid molecule. By introducing the vectorcomprising the nucleic acid molecule of the invention into an antigenpresenting cell, the peptide or peptide derivative of the invention isexpressed, and a complex between an HLA and a peptide or peptidederivative of the present invention is presented on the surface of thecell. Thus obtained antigen presenting cell can effectively increase apeptide-specific CTL against tumor cells.

Examples of vectors in which the nucleic acid molecule according to theembodiments of the present invention is incorporated may include variousplasmid vectors and viral vectors such as adenovirus, adeno-associatedvirus, retrovirus and vaccinia virus vectors (Liu M, Acres B, Balloul JM, Bizouarne N, Paul S, Slos P, Squiban P. Gene-based vaccines andimmunotherapeutics. Proc. Natl. Acad. Sci. USA 2004; 101 Suppl.,14567-71). Methods for preparing vectors have been well known in the art(Molecular Cloning: A laboratory manual, 2nd ed. New York, Cold SpringHarbor Laboratory).

The vector of the present invention may be administered to a patient sothat the peptide or peptide derivative according to the embodiments ofthe present invention is expressed in antigen presenting cells in thebody of the patient. Alternatively, the vector is introduced ex vivo ina suitable cell, for example a dendritic cell derived from the patient,so that the cell expresses the peptide or peptide derivative of theinvention, and then the cell is returned to the patient. Those methodsare well known in the art (Hrouda D, Dalgleish A G. Gene therapy forprostate cancer. Gene Ther. 1996; 3:845-52).

The amount of the vector to be administered may vary depending on thecondition of the disease to be treated, the age and body weight of thepatient to be treated and the like, and may preferably be 0.1 μg-100 mg,more preferably 1 μg-50 mg as an amount of DNA. The vector may beadministered, for example, intravenously, subcutaneously, orintradermally.

Pharmaceutical Composition

The present invention further provides a pharmaceutical composition forcomprising the peptide or peptide derivative according to the embodimentof the present invention, the nucleic acid molecule encoding the saidpeptide or peptide derivatives, or the vector comprising said nucleicacid molecule. The pharmaceutical composition of the present inventionis useful for the treatment or prevention of cancer. Especially, PSF1 isassociated with regeneration of the cancer stem cell and so useful forthe treatment or the prevention of cancer, which is treatment-resistantto chemotherapy or radiation therapy, recurrent, or metastatic. Thepharmaceutical composition of the present invention may comprise onepeptide or peptide derivative of the present invention, or a combinationof two or more peptides and/or peptide derivatives. Since a cancerpatient has a mixture of CTLs recognizing a plurality of differentcancer antigen peptides, it is effective to use a plurality of thepeptides or peptide derivatives of the present invention in combination.The peptide or peptide derivative of the invention may be used incombination with a cancer antigen peptide other than the peptide of thepresent invention.

The pharmaceutical composition of the present invention may comprise apharmaceutically acceptable carrier or the like in addition to thepeptide or peptide derivative. Examples of the carrier may includecellulose, amino acid polymers and albumin. The pharmaceuticalcomposition according to the embodiments of the present invention may beformulated as liposomal preparations, particulate preparations in whichthe ingredient is bound to beads having a diameter of severalmicrometers, or preparations in which the ingredient is attached tolipids. The pharmaceutical composition of the present invention may beadministered along with an adjuvant which has conventionally been usedfor vaccination in order to establish the immune response effectively.Suitable adjuvants are described in the literature (Johnson A G. Clin.Microbiol. Rev. 1994; 7:277-89). Exemplary adjuvants include, but arenot limited to, aluminum phosphate, aluminum hydroxide, and alum. Thecomposition may be administrated intradermally or subcutaneously.

The pharmaceutical composition of the present invention can be used as acancer vaccine. The dose may be determined based on the condition of thedisease to be treated, age and body weight of the respective patient.The amount of the peptide or peptide derivative, according to theembodiments of the present invention, in the pharmaceutical compositionmay be 0.0001 mg-1000 mg, preferably 0.001 mg-100 mg, more preferably0.01 mg-10 mg, even more preferably 0.1-5 or 0.5-30 mg. Thepharmaceutical composition may preferably be administered, every day,every week, or once every several days, several weeks or several monthsfor 1-3 years.

Method for Inducing CTL

The present invention further provides a method for inducing CTL.According to the embodiments of the present invention, the CTL hascytolytic effect against HLA-A02⁺ tumor cells. “Cytolytic” refers theproperty of the CTL which can recognize a complex between a cancerantigen peptide and an HLA on tumor cells and kill the tumor cells. ACTL according to the embodiments of the present invention can beobtained for example by incubating PBMCs collected from an HLA-A02⁺cancer patient in vitro in the presence of the peptide or peptidederivative of the present invention. The CTL induced by the presentmethod is useful for the adoptive immunotherapy wherein the induced CTLis returned into the patient from which the PBMCs have been collected tokill cancer cells.

Method for Inducing an Antigen Presenting Cell

Embodiments of the present invention further provide a method forinducing an antigen presenting cell. The method according to theembodiments of the present invention is a method for inducing an antigenpresenting cell which can induce a CTL against HLA-A02⁺ tumor cells. Themethod may be carried out, for example, by incubating a cell having anantigen-presenting ability derived from a HLA-A02⁺ cancer patient withthe peptide or peptide derivative of the present invention so that thepeptide or peptide derivative is bound to an HLA and presented on thecell surface. Alternatively, a vector which encodes the peptide orpeptide derivative of the present invention may be introduced into theaforementioned cell such that the peptide or peptide derivative isexpressed. The cell having an antigen presenting ability may be, forexample, dendritic cell. Dendritic cell derived from a patient can beprepared from PBMCs collected from the patient by separating cellsadhered to a culture plate and then incubating the separated cells inthe presence of IL-4 and GM-CSF for one week. The antigen presentingcell prepared by the method of the present invention can induce a CTLthat specifically recognizes a complex between a peptide or peptidederivative of the present invention and an HLA presented on the surfaceof the antigen presenting cell. When the antigen presenting cell of theinvention is administered to an HLA-A02⁺ cancer patient, they can inducea tumor-reactive CTL in the body of the patient.

Others

The present invention further provides a method for treating orpreventing tumor, which comprises administering the peptide, peptidederivative, or vector according to the embodiments of the presentinvention to a subject in need thereof. In addition, the presentinvention also provides use of the peptide, peptide derivative or vectorof the present invention for the manufacture of a pharmaceuticalcomposition for the treatment or prevention of tumor.

EXAMPLE

The present invention is further illustrated by the following examples,but is not restricted by these examples in any way.

Example 1: Direct Epitope Discovery

A cellular cytotoxicity T-cell (CTL) derived by a cancer vaccine isconsidered to recognize a complex of HLA-A02 molecule expressed on thesurface of tumor cells and peptide and attack the complex. Thus,proteins, from which peptides are derived, can be cancer antigens. Inview of this, a search was conducted for PSF1 sequence peptides capableof binding to HLA-A02 molecules of tumor cells so as to estimate thepossibility that PSF1 becomes cancer antigens.

(Method)

A direct epitope discovery method was performed under conditions withreference to a known report (Hawkins et al. J. Proteome. Res. 2008;7:1445-1457).

Organization of Soluble HLA Vector and Production of Stable Cell Line

An expression vector in which a CII tag (collagen type II) sequence wasbound to genes coding an extracellular protein region of a human HLA-A02sequence was organized and introduced to a human mammary carcinomastrain (MDA-MB-231, ATCC), thereby producing a stable cell line.

Purification and Identification of PSF1 Epitope Peptide

A conditioned medium of cells or cells obtained by transient forcedexpression of human PSF1 was collected, and the expressed HLA-A02molecule was purified with an anti-CII tag antibody column (seeInternational Publication Gazette WO2011/034128). The anti-CII tagantibody column was washed with 150 mM of a NaCl/20 mM Tris buffer and400 mM of a NaCl/20 mM Tris buffer, and then peptides binding to HLA-A02molecules were extracted with 10% acetic acid. The extracted peptideswere ultrafiltered with an Amicon Ultra 10K (Millipore Co., Temecula,Calif.), and fractionated with an SCX (GL Sciences Inc., Torrence,Calif.). Each fraction was subjected to peptide purification with a C18column (GL Sciences Inc., Torrence, Calif.) and then to centrifugalconcentration with a Speed Vac. A sample subjected to the centrifugalconcentration was dissolved in 0.1% TFA/2% acetonitrile again, measuredwith an LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Carlsbad,Calif.), and then was searched for peptide sequence derived from PSF1with a MASCOT algorithm (Matrix Science Ltd., London, United Kingdom).

(Results)

Five peptides having the same amino acid sequence (SEQ ID NO: 1) as thatof PSF1 were found. Table 1 shows the results. An HLA-A02 moleculepeptide complex collected from tumor cells included PSF1 peptides, andthus, the possibility that PSF1 serves as cancer antigens was found.Some identified peptides showed the HLA-A02 binding rule (Nature 1991;351:290-296), and thus, these peptides were supposed to have CTLinducing activity as cancer vaccine peptides.

TABLE 1 Initiating amino acid number Peptide sequence SEQ ID NO. 21LPAFNEDGLRQV 10 35 EMKALYEQN 11 79 YLYDRLLRI 3 88 RALRWEYGSVLPN 4 130GDEGLDITQDMKP 12

Example 2: In Silico Prediction

In Example 1, it was confirmed that PSF1 are cancer antigens, and aplurality of cancer vaccine candidate peptides were identified. On theother hand, in reverse immunology, peptides that can be bonded toHLA-A02 molecules are obtained by using software (Immunology and CellBiology 2006; 84:318-330). Thus, to obtain PSF1 peptide candidatesexcept those found in Example 1, a software search was carried out.

(Method)

Peptides that can be bonded to HLA-A02 molecules were predicted by usingthree types of software:

-   -   NetMHC <http://www.cbs.dtu.dk/services/NetMHC>    -   BIMAS <http://www-bimas.cit.nih.gov/molbio/hla_bind/>    -   SYFPEITHI <http://www.syfpeithi.de/>        To these types of software, a PSF1 full-length amino acid        sequence was input, and binding scores regarding peptides of 9        mer and 10 mer were obtained.        (Results)

For peptides of 9 mer and 10 mer derived from full-length PSF1, aplurality of peptides having high scores were obtained. Table 2 showsthe scores obtained by using SYFPEITHI (SEQ ID No.: 33). Table 2suggests the possibility that these peptides bind to HLA-A02 molecules.

TABLE 2 Initiating Sequence SYFPEITHI amino acid no. score SEQ ID NO. 5KAMELIREL 25 13 8 ELIRELHRA 18 14 31 QVLEEMKAL 18 15 35 EMKALYEQN 5 1169 LLRNRRCTV 24 16 79 YLYDRLLRI 27 3 89 ALRWEYGSV 24 17 120 SLATYMRSL 2718 145 SLYIEVRCL 27 19 159 FEVDDGTSV 14 20 38 ALYEQNQDSV 25 21 68SLLRNRRCTV 26 22 78 AYLYDRLLRI 16 6 79 YLYDRLLRIR 18 7 88 RALRWEYGSV 189 89 ALRWEYGSVL 22 8 97 VLPNALRFHM 15 23 127 SLGGDEGLDI 23 24 152CLKDYGEFEV 22 25 176 FLPRWKCEQL 21 26

Example 3: Mouse CTL Induction Assay (ELISPOT Assay)

It was determined whether the PSF1 peptides obtained by Examples aboveinduce peptide specific CTL through HLA-A02.

(Method)

Peptide Immunization and Collection of Responder Cells

The mouse CTL inducing activity with peptides was determined by usingmouse immune models. Regarding mice, CB6F1-Tg (HLA-A0201/H2-Kb)A0201 wasobtained from Taconic Biosciences, Inc. At most five types of peptidesolutions dissolved in DMSO were mixed to produce a PBS solution, andthe PBS solution was mixed together with the same amount of MONTANIDEISA 51VG (SEPPIC), thereby producing an emulsion antigen solution. Then,50 μL of the antigen solution was used for hypodermic injection intoeach of two sites at both sides of the belly of the mice (50 μgpeptide/mice, N=3-4). Two weeks after the injection, spleen was removed,and spleen cells from which erythrocyte had been eliminated with asolution (BD Pharm Lyse™, Becton, Dickinson and Company, La Jolla,Calif.) were collected as responder cells for an ELISPOT assay.

Production of Stimulator Cells

T2 cells (ATCC) of stimulator cells for use in the ELISPOT assay wereproduced by incubating, for one night, in an AIM-V culture mediumsupplemented with immunized assay peptide (30 μg/mL) or negative controlpeptide (ELAGIGILTV (SEQ ID NO.: 34)).

ELISPOT Assay

The ELISPOT assay was conducted by detecting IFN-γ produced fromresponder cells. The procedure thereof conformed to an accompanyingdocument of an assay kit, Mouse IFN-γ ELISpot^(PLUS) (MABTECH,Cincinnati, Ohio). Preincubation for the IFN-γ production was performedby adding cells to a 96-well plate included in the assay kit. Stimulatorcells were inoculated in a duplicate well or a single well of 5×10⁴cell/100 μL/well, and then responder cells were added to 2×10⁶ cell/100μL/well. After one-night coculture, the cells were removed and the wellwas washed, and then primary antibodies (biotin-antimouse IFN-γantibodies) included in the assay kit were added. After reaction for twohours at room temperature, the well was washed, supplemented withsecondary antibodies (streptavidin-ALP), and subjected to reaction forone hour at room temperature. After having been sufficiently washed, thecells were supplemented with a substrate solution and left for oneminute at room temperature. To terminate coloring reaction, the platewas sufficiently washed with running water and was air-dried.

Measurement of the Number of Spots and Measurement Method

The number of blue spots observed at the well bottom was measured withan ImmunoSpot® S5 Verse Analyzer (Cellular Technology Limited, ShakerHeights, Ohio). Measurement values of unclear spots because of a widerange of coloring were not used. An average value was obtained for theduplicate well, and the difference from the negative control wascalculated in order to obtain the number of spots specific to peptides.The average value and the SD value were calculated by using values forindividual mice, and the case having a positive number of differencesbetween the average value and the SD value was determined as positive.

(Results)

Peptide specific spots were observed in peptides shown in Table 3, and10 of these peptides were determined to be positive. Since specific CTLwere induced by PSF1 derived peptides, it was confirmed that peptideswere presented by HLA-A02, and mouse CTL recognizing PSF1 derivedpeptides was induced. Although the results were obtained for a mouseimmune system, the reaction occurs through HLA-A02, which is human MHC,and thus, it was suggested that there is the possibility of similarinduction of CTL in humans.

TABLE 3 Initiating Initiating amino amino acid Specific acid Specificnumber/ spot number/ spot SEQ ID number SEQ ID number NO. Sequence(Ave ± SD) NO. Sequence (Ave ± SD) 9 mer 10 mer   5/13 KAMELIREL 22 ± 13 38/21 ALYEQNQDSV  30 ± 15  31/15 QVLEEMKAL 7 ± 7  68/22 SLLRNRRCTV  1 ±2  35/11 EMKALYEQN 6 ± 8  78/6 AYLYDRLLRI  23 ± 21  69/27 LIRNRRCTV 7 ±9  79/7 YLYDRLLRIR 165 ± 17  79/3 YLYDRLLRI 80 ± 18  97/23 VLPNALRFHM127 ± 41 120/18 SLATYMRSL 70 ± 79 152/25 CLKDYGEFEV  53 ± 28 145/19SLYIEVRCL 43 ± 24 176/26 FLPRWKCEQL  31 ± 24  21/10 LPAFNEDGLRQV  3 ± 8 88/28 RALRWEYGSVLPN  29 ± 22 130/12 GDEGLDITQDMKP  1 ± 2

Example 4: Human CTL Induction Assay (ELISPOT Assay)

It was determined whether the PSF1 peptides obtained in Examples 1 and 2induce peptide specific human CTL.

(Method)

A human CTL induction assay was conducted under conditions withreference to a known report (Harano et al. Int. J. Cancer 2008;123:2616-2625).

Collection of Peripheral Blood Mononuclear Cells

Heparinized peripheral blood of healthy volunteers having HLA-A0201 wassubjected to centrifugation at 3000 rpm for 20 minutes, thereby removingplasma. Cell pellets were supplemented with HBSS(−) (Wako Pure ChemicalIndustries, Ltd, Richmond, Va.) to which 5 mM of HEPES (SIGMA, Carlsbad,Calif.) had been added, and were suspended, thereby overlaying thesuspension over a Ficoll-paque PREMIUM (GE Healthcare, Ltd., Pittsburgh,Pa.). Then, 400 g of the resulting substance was subjected tocentrifugation for 40 minutes, and peripheral blood monocyte cells(PBMCs) separated as an intermediate layer were collected.

Preparation of CD14-Positive Cells and CD8-Positive Cells

PBMCs were supplemented with CD14 microbeads (Miltenyi Biotec K.K.), andcaused to react at 4° C. for 15 minutes. The cells were passed throughLS columns (Miltenyi Biotec K.K., Tokyo, Japan), and CD14-positive cellswere obtained by using QuadroMACS™ Separator (Miltenyi Biotec K.K.,Tokyo, Japan). The rest of the cells were collected and washed, and thensupplemented with CD8 microbeads (Miltenyi Biotec K.K., Tokyo, Japan)and allowed to react at 4° C. for 15 minutes. Thereafter, a similarprocess is performed, thereby obtaining CD8-positive cells. TheCD14-positive cells were subjected to differentiation induction tomonocyte-derived dendritic cells (Mo-DCs), and CD8-positive cells weretemporarily frozen for storage.

Differentiation Induction to Mo-DC and Production of Peptide-PresentingCells

Since CD8-positive cells were subjected to antigen stimulation twice,peptide-presenting cells were produced twice accordingly. CD14-positivecells were disseminated in two RepCell plates (CellSeed Inc., Tokyo,Japan), and incubated in a 5% CO₂ incubator at 37° C. with a culturesolution in which 100 ng/ml GM-CSF (R&D Systems, Minneapolis, Minn.) and10 ng/ml IL-4 (R&D Systems) were added to an AIM-V (InvitrogenCorporation, Carlsbad, Calif.) culture medium including antibiotic andserum.

First production of peptide-presenting cells was started 5-7 days afterthe start of incubation. To one of the plates, 0.1 KE/ml OK-432 (ChugaiPharmaceutical Co., Ltd., Tokyo, Japan) was added, and on the next day,cells were collected and inoculated in a 96-well U-bottom plate. Then,20 μg/ml of assay peptide of one type was added to each half area (48wells) of the plate, and incubated for one day at 37° C. in a 5% CO₂incubator, thereby producing peptide-presenting cells. Second productionwas performed similarly on the other plate 12-14 days after the start ofincubation.

CTL Induction by Antigen Stimulation Using Peptide-Presenting Cells andPreparation of Responder Cells

Peptide-presenting cells in a 96-well plate were irradiated with X rays(30 Gy), and the frozen stored CD8-positive cells were added to all thewells, thereby performing first antigen stimulation. As a culturesolution, a solution in which 10 ng/ml IL-7 (R&D Systems, Minneapolis,Minn.) was added to an AIM-V culture medium including antibiotic andserum was used. After incubation for seven days, cells included insupernatant were collected and cocultured with peptide-presenting cells(after X-ray irradiation) obtained by the second production, therebyperforming second antigen stimulation in the 96-well plate. At thistime, 20 U/mL of IL-2 (Shionogi & Co., Ltd., Florham Park, N.J.) wasadded to the culture solution. After further incubation for seven days,cells in supernatant including CTL were collected and washed with anAIM-V culture medium including antibiotic and serum to have aconcentration of approximately 2×10⁵ cells/ml per well, therebyobtaining responder cells for an ELISPOT assay.

Production of Stimulator Cells

T2 cells of stimulator cells for use in an ELISPOT assay were producedby incubating the cells for one night in an AIM-V culture mediumsupplemented with an assay peptide (20 μg/mL). As stimulator cells of anegative control, only T2 cells were used (without peptides). The cellswere irradiated with X rays (30 Gy) and washed with an AIM-V culturemedium including antibiotic and serum to have a concentration of 2×10⁵cells/ml.

ELISPOT Assay

An ELISPOT assay was conducted by detecting IFN-γ produced fromresponder cells. The procedure thereof conformed to an accompanyingdocument of an assay kit, ELISpotPRO for Human IFN-γ (MABTECH,Cincinnati, Ohio). Responder cells in an amount of 100 μL/well wereadded for each 2 wells to a 96-well plate of the assay kit, and 100μL/well of stimulator cells supplemented with assay peptides orstimulator cells (without peptides) of a negative control wereinoculated in each well. After incubation for one night, the wells werewashed so that the cells were removed from the wells, and ALP-labelinganti-IFN-γ antibodies included in the assay kit were added. Afterreaction at room temperature for two hours, the cells were sufficientlywashed, supplemented with a substrate solution, and left at roomtemperature for five minutes. To terminate coloring reaction, the platewas sufficiently washed with running water and was air-dried.

Measurement of the Number of Spots and Measurement Method

The number of blue spots observed at the well bottom was measured withan ImmunoSpot® S5 Verse Analyzer. In a well including the same respondercells, the number of peptide specific spots was calculated bysubtracting the number of spots caused by stimulator cells (withoutpeptides) of a negative control from the number of spots caused bypeptide-added stimulator cells. Assuming that cells showing a differenceof 50 or more were defined to be positive, the following seven peptideswere determined to be positive in the examination on a plurality ofdonors.

(Results)

Human CTL in which seven peptides shown in Table 4 below were specificwas induced. Since specific CTL was induced by PSF1-derived peptides, itwas found that CTL recognizing PSF1 derived peptides exist in humans.This suggests the possibility that these PSF1 peptides can be a cancervaccine.

ELISPOT Assay Results

TABLE 4 Initi- Posi- ating tive SEQ Donor peptide donor ID Donorpositive number Sequence number NO. number percentage 21 LPAFNEDGLRQV 029 7   0% 35 EMKALYEQN 1 11 8  13% 79 YLYDRLLRI 6 3 13  46% 145SLYIEVRCL 2 30 2 100% 88 RALRWEYGSVLPN 1 4 8  13% 88 RALRWEYGSV 3 9 5 60% 89 ALRWEYGSVL 2 8 2 100% 89 ALRWEYGSV 2 17 5  40% 130 GDEGLDITQDMKP0 12 7   0%

Example 5: Human CTL Induction Assay (Tetramer Assay)

To confirm induction of PSF1 peptide specific CTL with another method, aTetramer assay was conducted.

(Method)

Production of CTL Line

Cells that had been determined to be positive in the above-describedELISPOT assay was continued to be incubated with a culture solution inwhich 100 ng/ml of IL-15 (Miltenyi Biotec K.K., San Diego, Calif.) wasadded to an AIM-V culture medium including antibiotic and serum.

Tetramer Assay

Tetramer (Medical & Biological Laboratories Co., Ltd: MBL) for PSF1peptides (YLYDRLLR, SEQ ID NO.: 31) was produced. Cells of a CTL linewere collected, colored with tetramer and APC H7 marker anti-CD8antibodies (SK-1) (Becton, Dickinson and Company, La Jolla, Calif.), andmeasured with a FACS Aria (Becton, Dickinson and CompanyLa Jolla,Calif.). Two types of CTL lines (0209-01 H2 and 0209-02 D2) were used.

(Results)

As shown in FIG. 1, since a cell population that reacts with bothanti-CD8 antibodies and tetramer was observed in each CTL line, it wasfound that CTL lines recognizing PSF1 peptides were produced.Observation of Tetramer-positive cells directly proved the presence ofpeptide-specific human CTL.

Example 6: Human CTL Cytotoxic Assay

To examine whether a CTL line has an activity of attacking cells, acytotoxic assay was conducted.

Specifically, assay conditions were set with reference to a methoddescribed in a known report (New CFSE-based assay to determinesusceptibility to lysis by cytotoxic T cells of leukemic precursor cellswithin a heterogeneous target cell population. Jedema I. et al. Blood2004; 103:2677-2682).

Production of Target Cells

As target cells for use in the cytotoxic assay, 1 μM of CellTracker™Green CMFDA (5-chloromethylfluorescein diacetate) (Invitrogen Corp.,Carlsbad, Calif.) was added to T2 cells, and these cells were allowed toreact with each other at 37° C. for 15 minutes. The T2 cells werelabeled with CMFDA, which is a fluorescent marker, washed with AIM-V,supplemented with assay peptides (20 μg/ml), and incubated for onenight. As target cells of a negative control, T2 cells only labeled witha fluorescent marker were used (without peptide addition). The cellswere washed with an AIM-V culture medium including antibiotic and serumto have a concentration of 5×10⁴ cells/ml.

Production of Effector Cells

For effector cells for use in the cytotoxic assay, three types of CTLlines (0209-1 H2; YLYDRLLRI (SEQ ID NO.: 3), 1004-1 P-13-1_2;RALRWEYGSVLPN (SEQ ID NO.: 4), and 1004-1 P-10-9_1; ALRWEYGSVL (SEQ IDNO.: 32)) obtained by incubation after the above-described ELISPOT assaywere used. The cells were prepared to have concentrations of 50, 15,5×10⁴ cells/ml after washing with AIM-V. The preparation was performedwith an AIM-V culture medium including antibiotic and serum.

Cytotoxic Assay

Target cells (T) supplemented with peptides used in CTL induction ortarget cells (T) of a negative control supplemented with no peptideswere mixed with effector cells (E) at a cell ratio (E/T ratio) of 10:1,3:1, and 1:1, and cocultured for one night. Immediately beforemeasurement with a MACS Quant (Miltenyi Biotec, San Diego, Calif.) or aFACS Aria, 0.1 μg/ml of DAPI was added in order to distinguish livingcells from dead cells by means of staining. In an analysis, theproportion of DAPI-negative cells (living cells) in target cells labeledwith CMFDA was obtained, and the survival rate of target cells wascalculated using incubation conditions (only for target cells) wherecoculture with effector cells was not performed as 100%.

(Results)

As shown in FIG. 2, in the assay on each of the CTL lines, the survivalrate of target cells supplemented with peptides was lower than that oftarget cells of a negative control (without peptides). It was found thatthe three types of CTL lines induced by peptides showed specificrecognition of peptides and show cytotoxic effect. This suggests thepossibility that tumor cells presenting PSF1 peptides on the cellsurfaces are damaged by CTL recognizing PSF1 peptides, and thus,suggests that PSF1 peptides can be a cancer vaccine.

The present invention provides a peptide inducing CTL significantly, aDNA encoding of said peptide, a pharmaceutical composition comprisingsaid peptide and said DNA, and a cancer vaccine using said peptide andsaid DNA.

While the principles of the disclosure have been described above inconnection with specific examples, it is to be clearly understood thatthis description is made only by way of example and not as limitation onthe scope of the invention.

The invention claimed is:
 1. A method of inducing cytotoxic T-cellactivity in a cell comprising the steps of: administering a peptideselected from the group consisting of the amino acid sequences of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, wherein at least one of the amino acids of saidamino acid sequence optionally consists of N-terminal acylation orC-terminal amidation, and the peptide has cytotoxic T-cell inducibilityto the cell.
 2. The method according to claim 1, wherein the said aminoacid sequence includes a formyl group, an acetyl group, or at-butoxycarbonyl group at the N-terminus of the peptide.
 3. The methodaccording to claim 1, wherein the said amino acid sequence includes amethyl group, an ethyl group, a t-butyl or a benzyl group on theC-terminus of the peptide.
 4. The method according to claim 1, whereinthe cell is an antigen presenting cell.
 5. The method according to claim4, wherein the antigen presenting cell is obtained from a Humanleucocyte Antigen-A02 (HLA-A-A02) positive (HLA-A02⁺) cancer patient. 6.The method according to claim 5, wherein a cancer cell obtained from thecancer patient expresses Partner of Sld five 1 (PSF 1) on its surface.